PROJECT SUMMARY Determining how neurons are correctly specified and assembled into functional circuits will provide insight into developmental disorders of the nervous system and may suggest therapeutic approaches to promote nerve regeneration. Slit and Netrin, and their Robo and Fra/DCC receptors, are evolutionary conserved families of signaling molecules that play important roles in regulating neuronal development; however, the understanding of how these receptors are regulated and how they signal to direct axon growth and guidance is incomplete. These are important questions because perturbations of these signaling pathways are implicated in diseases of nervous system development. Slits, Netrins and their receptors also play essential roles outside of the nervous system and disruptions of these pathways are associated with several kinds of cancer. Our research program focuses on three broad areas related to the roles and regulation of these molecules in neuronal development using the genetically tractable Drosophila embryonic nervous system as a model. First, we are working to define functional and molecular links between conserved transcriptional regulators that impart neuronal subtype identity and the Robo and Fra/DCC receptors that coordinate axon guidance and dendrite morphogenesis in response to Slit and Netrin. Here, we will use genetic and molecular screening approaches, including Fluorescence Activated Cell sorting (FACs) of defined subsets of motor neurons, together with transcript profiling in wild type and mutant backgrounds, to systematically identify additional effectors of these transcriptional programs. Second, we are characterizing a newly discovered mechanism through which the Frazzled/DCC receptor intracellular domain (ICD) itself can act in the nucleus as a transcriptional activator to regulate commissureless expression to ensure that commissural axons avoid premature responses to midline repellent Slit. Here, we will use genetic and molecular approaches to identify factors that cooperate with the Fra ICD to regulate transcription and transcript profiling methods to identify additional targets of the Fra ICD. In addition, we will explore whether signaling from the nucleus is a common property of axon guidance receptors and through collaboration we will test if this is a conserved property of guidance receptors. Third, we are determining the molecular mechanisms underlying Robo and Fra/DCC receptor signaling during axon guidance using molecular, genetic, biochemical and cell biological approaches. Specifically, we use in vivo genetic manipulation, together with fluorescently tagged receptors and reporters of signaling molecule activity in vitro, to define the cell biological outputs of receptor activation with sub-cellular resolution. Our research program will define new concepts in the molecular biology of axon guidance, inform studies of related proteins in mammalian systems and will likely enhance our understanding of neural developmental disorders.